Golden2-like (GLK) nuclear transcription factors are known to regulate chloroplast biogenesis in leaf tissues of angiosperms and mosses (Yasumura et al., 2005, Plant Cell 17:
1894-1907; Waters and Langdale, 2009, EMBO J28: 2861-2873). While many advances have been made in understanding of leaf chloroplast biogenesis, the role of GLK transcription factors has not been characterized in fruit.
The green tomato fruit pericarp contains photosynthetically active chloroplasts (Piechulla et al., 1985, Plant Molecular Biology 5: 373-384; Piechulla et al., 1987, Plant Physiology 84: 911-917; Blanke and Lenz, 1989, Plant Cell and Environment 12: 31-46; Gillaspy et al., 1993, Plant Cell 5: 1439-1451; Carrara et al., 2001, Photosynthetica 39: 75-78) and it has been reported that as much as 20% of the total carbon in fruit is a consequence of photosynthetic activity in the green fruit itself. Proteins involved in light harvesting electron transfer and CO2 fixation are present in tomato fruit. While many components of the photosynthetic apparatus are conserved in leaves and fruits, key differences exist, suggesting possible fruit-specific regulation. The photosynthetic mechanisms that are operative in tomato fruit chloroplasts are not clearly defined but anatomical and metabolic differences suggest that fruit-specific mechanisms may be important (Blanke and Lenz, 1989; Hetherington et al., 1998, Journal of Experimental Botany 49: 1173-1181; Carrara et al., 2001). For example, many fruit, including tomatoes, lack or have very few stomates, so it is unclear how CO2 is assimilated and regulated in fruit. Tomato fruit can fix CO2 by utilizing ribulose 1,5-bisphosphate carboxylase (RuBPCO) and phosphoenolpyruvate carboxylase (PEPCase), but endogenous respiration in fruit make it difficult to measure net assimilation of CO2 and details of the mechanisms of tomato fruit photosynthesis has not been resolved (Blanke and Lenz, 1989; Hetherington et al., 1998). Chlorophyll, the intact photochemistry of Photosynthesis System 2 (PS2) and the presence of carbon assimilation enzymes suggest that green fruit pericarp chloroplasts contribute to the overall carbon and energy required for fruit development (Smillie et al., 1999, Journal of Experimental Botany 50: 707-718). Furthermore, tomato fruit that develop in the absence of light have rudimentary chloroplasts with little or no chlorophyll or thylakoid grana and these fruit exhibit reduced sugars when ripe. This suggests that fruit chloroplasts may contribute to the overall accumulation of sugars by the fruit.
Chloroplast biogenesis is a complex process that requires close co-ordination of plastid and nuclear genomes, and many proteins that accumulate in the chloroplast are encoded by the nuclear genes (Fitter et al., 2002, Plant Journal 31: 713-727). Expression of the Golden2-like (GLK) genes, members of the GARP family of MYB transcription factors, is known to be required for chloroplast biogenesis and maintenance in the vegetative tissues of maize, rice and Arabidopsis (Fitter et al., 2002; Waters and Langdale, 2009). As has been observed in other plants, Arabidopsis has two redundant GLK genes, AtGLK1 and AtGLK2. Mutations in AtGLK1 or AtGLK2 do not alter leaf chloroplast morphology, although the siliques (seed capsules) of the Atglk2 mutant are pale green. Atglk1Atglk2 double mutants have attenuated chloroplast development and all of the leaves of the plants are light green. AtGLK1 regulates photosynthesis in specific cell types (Waters et al., 2008, Plant Journal 56: 432-444). In maize, ZmGLK1 and ZmGLK2 (G2) are associated with C3 photosynthesis, but ZmGLK1 is responsible for differentiation in C4 mesophyll cells and ZmGLK2 functions in C4 bundle sheath cells, suggesting cell-type specific regulation of C4 photosynthetic capacity by these transcription factors (Rossini et al., 2001, Plant Cell 13: 1231-1244). Partial complementation of the Atglk1 Atglk2 double mutant by the moss Physcomitrella patens PpGLK1 gene indicates that GLKs are similar in bryophytes and vascular plants, although because AtGLK1 is unable to complement a Ppglk1 Ppglk2 double mutant, some aspects of the function and/or regulation of GLKs may be species specific.
While the functions of GLK transcription factors have been inferred by examining leaf phenotypes, their role in fleshy green fruit development has not been evaluated. In particular, the art fails to show a link between GLK activity and fruit traits such as starch, soluble solids and/or sugars. The present invention addresses these and other needs.